Surface plasmon resonance for real-time monitoring of molecular interactions between a triple helix forming oligonucleotide and the Sp1 binding sites of human Ha-ras promoter: effects of the DNA-binding drug chromomycin.
DNA-binding molecules have been recently proposed as potential inhibitors of molecular interactions between transcription factors and target DNA sequences. Among DNA-binding drugs, chromomycin binds to GC-rich sequences of the Sp1 binding sites of the Ha-ras oncogene. These sites are also molecular targets of a triple-helix forming oligonucleotide [Sp1(Ha-ras)TFO] which is able to inhibit Ha-ras oncogene transcription. We studied molecular interactions between triple-helix forming oligonucleotides and target Sp1 binding sites of the human Ha-ras promoter in the presence of the DNA-binding drug chromomycin. This study was performed by (a) surface plasmon resonance and biosensor technology, (b) gel retardation assay and (c) magnetic capturing of molecular complexes between TFO, chromomycin and target DNA. The main conclusion of our study is that low concentrations of chromomycin allow binding of the triplex-forming oligonucleotide to Sp1 target DNA sequences of the Ha-ras oncogene promoter. Higher concentrations of this DNA-binding drug fully suppress molecular interactions between the Sp1(Ha-ras)TFO and target DNA. Additionally, low concentrations of chromomycin potentiate the effects of the Sp1(Ha-ras)TFO in inhibiting the molecular interactions between purified Sp1 transcription factor and target DNA sequences.
Rutigliano
C
C
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
Bianchi
N
N
Tomassetti
M
M
Pippo
L
L
Mischiati
C
C
Feriotto
G
G
Gambari
R
R
D001665:Binding Sites; D002273:Carcinogens; D002865:Chromomycins / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D011905:Genes, ras / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000187:drug effects* / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
D002273:Carcinogens; D002865:Chromomycins; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D016329:Sp1 Transcription Factor